Development of methods for precise modification of the human genome, to correct or eliminate disease alleles in the very cells they affect in patients, would have enormous impact on our approach to the treatment of many human diseases. We propose to lay the foundation for such therapies by testing key elements of treatment strategies designed to introduce specific changes into the rhodopsin gene in rod photoreceptor cells in mice. Dominant mutations in the rhodopsin gene cause the most common form of the most common hereditary blinding disease, retinitis pigmentosa (RP). This progressive neurodegenerative disorder begins with the death of rod photoreceptors, where rhodopsin is expressed, but ultimately destroys rod and cone cells, leading to loss of both dim-light and color vision. Successful modification of even a fraction of rod cells would likely extend the lifetime of useful vision, which could provide significant clinical benefit. To pursue these strategies, we have generated a set mouse lines that each carry a modified human rhodopsin-GFP fusion gene in place of the normal mouse rhodopsin gene to provide visible markers for gene modification. Our overarching hypothesis is that double-strand breaks (DSBs) targeted to specific sites in the rhodopsin gene can be used to correct or knockout the function of dominant rhodopsin mutations. To that end, we have constructed six zinc-finger nucleases (ZFNs) to cleave specific sites in the rhodopsin-GFP target genes, and packaged the ZFNs into recombinant adeno-associated virus (rAAV), which we will inject subretinally in mouse eyes. Our initial experiments demonstrate that efficient rhodopsin-gene cleavage and robust repair occur by both homologous recombination (HR) and nonhomologous end joining (NHEJ). We propose three Specific Aims designed to test various strategies for dealing with the dominant rhodopsin mutations that cause RP.
In Aim 1, we will test strategies for efficient correction of dominant mutations in the rhodopsin gene by HR.
In Aim 2, we will test strategies to efficiently knock out dominant mutations in the rhodopsin gene by NHEJ.
In Aim 3, we will test a general strategy for knocking out rhodopsin gene expression by modifying the gene to stimulate nonsense-mediated decay, thereby eliminating the mRNA. Overall, the proposed studies will test key elements of general treatment strategies designed to correct or eliminate dominant mutations that compromise the function of rod cells and ultimately cause their death. If successful, these approaches would provide general methods for treating the dominant rhodopsin mutations known to cause RP.

Public Health Relevance

Dominant mutations in the rhodopsin gene cause the most common form of the most common hereditary blinding disease, retinitis pigmentosa, which is a progressive neurodegenerative disorder that ultimately destroys rod and cone cells, leading to loss of both dim-light and color vision. We propose to deliver double-strand breaks to specific sites in the rhodopsin gene in mouse rods cells in order to correct or knockout the function of dominant rhodopsin mutations. If successful, these approaches would provide general methods for treating the dominant rhodopsin mutations that are known to cause retinitis pigmentosa.